Molecular basis for receptor recognition and broad host tropism for merbecovirus MjHKU4r-CoV-1

A novel pangolin-origin MERS-like coronavirus (CoV), MjHKU4r-CoV-1, was recently identified. It is closely related to bat HKU4-CoV, and is infectious in human organs and transgenic mice. MjHKU4r-CoV-1 uses the dipeptidyl peptidase 4 (DPP4 or CD26) receptor for virus entry and has a broad host tropism. However, the molecular mechanism of its receptor binding and determinants of host range are not yet clear. Herein, we determine the structure of the MjHKU4r-CoV-1 spike (S) protein receptor-binding domain (RBD) complexed with human CD26 (hCD26) to reveal the basis for its receptor binding. Measuring binding capacity toward multiple animal receptors for MjHKU4r-CoV-1, mutagenesis analyses, and homology modeling highlight that residue sites 291, 292, 294, 295, 336, and 344 of CD26 are the crucial host range determinants for MjHKU4r-CoV-1. These results broaden our understanding of this potentially high-risk virus and will help us prepare for possible outbreaks in the future.


Introduction
Coronaviruses (CoVs) belonging to the Coronaviridae family are enveloped, positive-sense, single-stranded RNA viruses that are classified into four genera: Alpha, Beta, Gamma, and Deltacoronavirus (Woo et al, 2012).Three BetaCoVs have been reported to be life-threatening and caused outbreaks, including severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003, the Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since 2020 (Allen et al, 2017;Jones et al, 2008;Sohrabi et al, 2020) which has posed major threats to public health and caused serious social and economic impacts.
Recurrent coronavirus zoonoses and the detection of numerous coronaviruses in wildlife suggest that cross-species transmission events are constantly occurring (Tortorici et al, 2022).SARS-CoV and SARS-CoV-2 are generally believed to originate from bats (Hu et al, 2017;Lu et al, 2020;Wang et al, 2021;Tan et al, 2020;Zhu et al, 2020).Bats are also suspected to be the natural reservoir of MERS-CoV (Wang et al, 2014;Memish et al, 2013;Ithete et al, 2013).However, the fact that no virus with a whole genome is highly homologous to MERS-CoV prevents concluding that MERS-CoV originated from bats (Wang et al, 2021;Cui et al, 2019).As the largest reservoir of Alpha and BetaCoVs, bats play pivotal roles in cross-species transmission of coronaviruses (Wang et al, 2014;Li et al, 2005a;Woo et al, 2012).However, rare physical contact between bats and humans occurs, and bat CoVs are normally required to evolve further to acquire the ability to infect humans, suggesting intermediate animal hosts may be needed (Chen et al, 2023;Zhao et al, 2022a).For a while, civets and dromedary camels were regarded as such intermediates for SARS-CoV and MERS-CoV, respectively (The Chinese SARS Molecular Epidemiology Consortium, 2004;Li et al, 2005b;Song et al, 2005;Guan et al, 2003;Haagmans et al, 2014;Reusken et al, 2013).However, this notion might need to be reassessed.Identification of two SARS-CoV-2-related coronaviruses, GD/1/2019 and GX/P2V/2017 (Lam et al, 2020;Xiao et al, 2020), suggests that pangolins have potential as intermediate animal hosts.A recent study isolated a pangolinorigin MERS-like CoV, MjHKU4r-CoV-1, that is related to bat HKU4-CoV but may be capable of directly infecting humans and exhibiting enhanced infectivity to human cells/organs compared to bat viruses (Chen et al, 2023).
The binding capacity of these receptors for viral S proteins is one of the crucial factors determining viral infectivity and pathogenicity.Previous studies displayed that CD26 interacts with the receptor-binding domain (RBD) of MERS-CoV, not the NTD (Lu et al, 2013;Yuan et al, 2020).Recently reported MjHKU4r-CoV-1 also uses its RBD to interact with CD26 for virus entry (Chen et al, 2023).Thus far, the molecular basis of CD26 recognition by MjHKU4r-CoV-1 remains unknown.Furthermore, previous data demonstrate that MjHKU4r-CoV-1 might have a broad host range (Chen et al, 2023).However, its underlying determinants were not demonstrated, hindering our understanding of the zoonotic risks of this virus.
In this study, we measure the binding affinity between human CD26 (hCD26) and the RBD of MjHKU4r-CoV-1, HKU4-CoV, or MERS-CoV and explore the molecular mechanism of MjHKU4r-CoV-1 RBD binding to hCD26.We then evaluate the receptor binding capability of the MjHKU4r-CoV-1 RBD for 17 other animal CD26 orthologs, covering eight orders, and further identify critical host range determinants by mutagenesis analyses and structural homology modeling.Altogether, our data delineate the molecular basis of receptor recognition and determinants of the broad host tropism of a novel merbecovirus, MjHKU4r-CoV-1, highlighting the importance of in-depth research on these potentially high-risk viruses and providing important molecular evidence for possible outbreaks in the future.

Structural comparisons of MjHKU4r-CoV-1, HKU4-CoV, and MERS-CoV RBDs in complex with hCD26
Previous work demonstrates the molecular mechanism of viral entry of HKU4-and MERS-CoVs using hCD26 (Wang et al, 2014;Lu et al, 2013).To reveal why MjHKU4r-CoV-1 has stronger binding affinities than HKU4-CoV and MERS-CoV, we analyzed the features in the interface between the RBDs and hCD26.Due to the AA distinctions among the MjHKU4r-CoV-1, HKU4-CoV, and MERS-CoV RBDs, the RBD/hCD26 interfaces display significant differences (Fig. 3A-C).For convenient structural comparison, the AA sequences of the HKU4-CoV and MERS-CoV RBDs were renumbered based on that of the MjHKU4r-CoV-1 RBD, as shown in the AA sequence alignment among the MjHKU4r-CoV-1, HKU4-CoV, and MERS-CoV RBDs (Fig. 1A).Structural comparisons revealed that although there are many shared key residue sites among these two or three RBDs, several shared sites are occupied by different AA residues (Fig. 3A-D), which may affect their interactions toward CD26.Among these, residues H517 and R550 of the MjHKU4r-CoV-1 RBD have the most evident contact increase than N517 and K550 of the HKU4-CoV RBD, showing 21 and 13 contact differences, respectively (Fig. 3A,B).Compared with the MERS-CoV RBD, residues H517 and S519 of MjHKU4r-CoV-1 RBD could contribute to higher binding capacity.In addition, there are many unique sites for each RBD to engage with hCD26 (Fig. 3D).Notably, the interface formed by residues S502, F537, S538, Y540, G541, F542 and F549 of the MjHKU4r-CoV-1 RBD  interacts with the N-glycans of N229 in hCD26, which is not observed in the HKU4-CoV RBD/hCD26 and MERS-CoV RBD/ hCD26 complexes due to the shorter N-glycans resulting from different protein expression systems.In this work, a mammalian expression system was used for hCD26 expression.However, the hCD26 protein for the HKU4-CoV RBD/hCD26 and MERS-CoV RBD/hCD26 complexes was expressed using the bac-to-bac baculovirus system, which produces simpler sugar modifications.Residue Y540 of the MjHKU4r-CoV-1 RBD has a longer side chain than G540 of the HKU4-CoV RBD and S540 of the MERS-CoV RBD (Fig. 1A), potentially contributing to a higher binding affinity toward N-glycans of N229 on hCD26.
The interaction area (excluding N-glycans) of hCD26 in the MjHKU4r-CoV-1 RBD/hCD26 complex is smaller than that of the HKU4-CoV RBD/hCD26 and MERS-CoV RBD/hCD26 complexes (Fig. 3E), and contains the fewest van der Waals contacts (VDWs).However, the structural alignment shows that the MjHKU4r-CoV-1 RBD is closer to hCD26 and its N229 N-glycans than the HKU4-CoV and MERS-CoV RBDs (Fig. EV2), suggesting that the MjHKU4r-CoV-1 RBD forms stronger interactions to bind more tightly to hCD26 than the other two RBDs.
K509 in the RBD is conserved in the three complexes and forms two H-bonds with T288 and A289 of hCD26 (Fig. 4B).R550 in the MjHKU4r-CoV-1 and MERS-CoV RBDs interacts with L294 of hCD26 (Fig. 4B), while residue 550 is K in the HKU4-CoV RBD and forms an H-bond with I295, not L294 (Fig. 4B).Notably, residue N508 of the MERS-CoV RBD is hydrogen-bonded with Q286 of hCD26, which is not observed in the other two complexes (Fig. 4B).
D545 in the MjHKU4r-CoV-1, HKU4-CoV, and MERS-CoV RBDs forms an H-bond with K267 of hCD26 (Fig. 4C).K267 of hCD26 also forms an extra H-bond with E544 of the HKU4-CoV RBD or D547 of MERS-CoV RBD (Fig. 4C).Residue 547 of the HKU4-CoV RBD (Q547) or MjHKU4r-CoV-1 (E547) differs from that of the MERS-CoV RBD (D547) (Fig. 4C).Q547 of the HKU4-CoV RBD engages with N-glycans of N229 together with E544 of the RBD, while E547 of the MjHKU4r-CoV-1 RBD interacts with N-glycans of N229 together with S543 of the RBD (Fig. 4C).Notably, no residues of the MERS-CoV RBD were observed to form H-bonds with the N-glycans of N229 on CD26.In contrast, there is an additional H-bond between T265 of hCD26 and Y548 of the MERS-CoV RBD (Fig. 4C).
The underlined numbers suggest the number of potential H-bonds between the pairs of residues.VDWs were analyzed at a cutoff of 4.5 Å, and H-bonds were calculated at a cutoff of 3.5 Å.
To decipher how these determinant AAs impact the binding capacity of CD26s from Tp bat, rat, hamster, cat, dog, and ferret for the MjHKU4r-CoV-1 RBD, homology modeling was performed (Fig. 7A,B).All models from the above six species displayed high similarity to hCD26, with a root mean square deviation (RMSD) range of 0.108 to 0.131.Among the six structures, high coverage ranging from 99-100% and high model quality estimation, including Global Model Quality Estimation (GMQE), global absolute quality estimates on the basis of one single model (QMEANDisCo Global), and Quaternary Structure Quality Estimation (QSQE) (Table 4), indicated a lack of major conformational changes between species and supported the validity of using hCD26 as a template for modeling these CD26 interface residues across species.AA sequence alignment showed that residue 295 in the CD26 from human, macaque, marmoset, rabbit, Malayan pangolin, horse, goat, sheep, pig, cattle, and camel is the hydrophobic residue isoleucine (I).However, residue 295 is the hydrophilic residue K in the Tp bat CD26 and threonine (T) in the CD26 from Pp bat, rat, hamster, cat, dog, and ferret (Fig. 6A).In our MjHKU4r-CoV-1 RBD/hCD26 structure, I295 forms extensive hydrophobic interactions with V513, I561, and V563 of the MjHKU4r-CoV-1 RBD (Fig. 1E).I295K or I295T substitutions would disrupt these hydrophobic interactions, and residue K has a longer side chain, forming steric clashes with the MjHKU4r-CoV-1 RBD, both of which lead to the reduction even ablation of the binding capacity for Mj-RBD-CoV-1, consistent with the SPR results (Figs. 6B, 7A and EV3, Table 3).Similarly, T294 and S294 observed in the rat and ferret CD26s would also impair the hydrophobic interactions with residues V548, V563, and V565 of the MjHKU4r-CoV-1 RBD, and T294 may have steric clashes with the MjHKU4r-CoV-1 RBD, thereby abolishing binding (Figs. 6B, 7A and EV3, Table 3).In addition, residue 291 in the CD26 from hamster, dog, and ferret is E291, P291, and D291, respectively.Both E291 and D291 have a longer side chain that forms steric hindrance with the MjHKU4r-CoV-1 RBD to abrogate binding (Figs. 6B and 7A, Table 3).Although residue P291 has little impact on the dog CD26 for binding to the MjHKU4r-CoV-1 RBD, its neighboring residue D292 forms severe steric clashes to block binding to the MjHKU4r-CoV-1 RBD (Figs. 6B and 7A, Table 3).By contrast, A292 has a somewhat shorter side chain than S292, which slightly decreases the binding capacity (Fig. 7A and EV3, Table 3).
The side chain of R336 in hCD26 forms an H-bond with Y463 and many VDWs with Y463, D471, and Y506 of the MjHKU4r-CoV-1 RBD (Fig. 1D, Table 2).V336 in the rat CD26, T336 in the hamster CD26, K336 in the cat CD26, G336 in the dog CD26, and S336 in the ferret CD26 all have a shorter side chain, which could decrease the binding capacity for the MjHKU4r RBD (Fig. 7B), consistent with the SPR results (Fig. EV3, Table 3).Notably, Q344 of hCD26 is hydrogen-bonded with E521 in the MjHKU4r-CoV-1 RBD/hCD26 complex (Fig. 7B).E344 in the CD26s from rat, cat, (A-C) The binding interface of HKU4-CoV RBD/hCD26 (PDB: 4QZV) (Wang et al, 2014) (A) MjHKU4r-CoV-1 RBD/hCD26 (B), and MERS-CoV RBD/hCD26 (PDB: 4KR0) (Lu et al, 2013) (C).Three RBDs and hCD26 are shown as surfaces, and interaction areas are colored in cyan (HKU4-CoV RBD), hot pink (MjHKU4r-CoV-1 RBD), pink (MERS-CoV RBD) and orange (hCD26), respectively.The AA sequences of HKU4-CoV and MERS-CoV RBDs are renumbered based on the MjHKU4r-CoV-1 RBD for convenient structural comparison.Interacting residues on the HKU4-CoV, MjHKU4r-CoV-1, and MERS-CoV RBDs are labeled, among which only the same AA residues shared by these three RBDs are colored in black; otherwise in cyan, hot pink, and pink, respectively.Similarly, interacting residues on hCD26 shared by three complexes are labeled in black, otherwise in orange.The numbers of the VDWs for each residue are in parentheses.(D) Venn diagrams of interacting residues on the HKU4-CoV, MjHKU4r-CoV-1, and MERS-CoV RBDs for hCD26.Residues involved in forming H-bonds or hydrophobic interactions with hCD26 and shared by three RBDs are marked in bold.Residues that potentially confer MjHKU4r-CoV-1 RBD with a higher binding affinity are highlighted with black boxes.(E) Venn diagrams of residues on hCD26 bound to the HKU4-CoV, MjHKU4r-CoV-1, and MERS-CoV RBDs.Residues tested in Fig. 2 are marked in bold, and key residues among them are further highlighted with black boxes.and ferret may change the surface electrostatic properties of CD26 to repulse E521 of MjHKU4r-CoV-1 RBD to push themselves away from the interface, thus decreasing the binding affinity between CD26 and MjHKU4r-CoV-1 RBD, as observed in the SPR results (Fig. 6B and EV3, Table 3).

Discussion
Carbohydrates play pivotal roles in molecule interactions.Like other merbecoviruses (Wang et al, 2014;Lu et al, 2013), the MjHKU4r-CoV-1 S protein interacts with N-glycans on the hCD26 receptor.AA sequence alignment shows that these N-glycans, located at N229 on hCD26, are conserved among animal species.Our previous work showed that HKU4-CoV, utilizing the bat CD26, also needs N-glycan binding from the bat CD26 (Yuan et al, 2020).We assessed the mutations of N229 to A229 or Q229 to evaluate the N-glycans effect on the binding affinity.Unfortunately, hCD26 mutant proteins harboring A229 or Q229 were not successfully expressed, implying that the loss of N-glycans of N229 may adversely affect the expression, folding, or stability of hCD26.
The K D values for MjHKU4r-CoV-1 RBD binding to hCD26 reported in Fig. 1 (0.41 ± 0.03 μM), 2 (0.36 ± 0.04 μM), 5 (0.26 ± 0.04 μM), and 6 (0.23 ± 0.09 μM) are different, which may be caused by variability inherent to protein activity differences among different protein preparations, protein quantification by the bicinchoninic acid assay, and SPR measurements.Furthermore, the SPR results for the MjHKU4r-CoV-1, HKU4-CoV, and MERS-CoV binding to hCD26 displayed some differences from previous studies.The binding affinity between the MjHKU4r-CoV-1 RBD and hCD26 that we measured (0.41 μM) is higher than that of a previous study (3.25 µM), as shown for the binding affinity between the HKU4-CoV RBD and hCD26 (5.62 µM vs. very weak) (Chen et al, 2023).In other reports, the affinity of the HKU4-CoV RBD for hCD26 is ~35.7 µM (Wang et al, 2014), which could be caused by the differences in measurement methods (SPR vs. biolayer interferometry, amine coupling vs. protein biotinylation, and CD26 immobilized and RBD flowed over vs. RBD immobilized and the CD26 flowed over).Given that CD26 was purified as a homodimer and RBD was purified as a monomer, the mode in which CD26 acts as the ligand and RBD as an analyte flowed over would be more suitable for 1:1 binding than the opposite way.Furthermore, to exclude potential interference with the functionally relevant part of the interacting molecule caused by amine coupling or protein biotinylation, a Twin-Strep-tag Capture Kit was used to capture hCD26 harboring a Twin-Strep tag at its C-terminus, and then the MjHKU4r-CoV-1 RBD with a His tag at its C-terminus was flowed over the sensor chip.The SPR results showed that the binding affinity between hCD26 and the MjHKU4r-CoV-1 RBD measured by this capture method (Fig. EV4) is similar to that measured from the amino coupling (Figs.1B, 2A, 5 and 6B).In our results, the binding affinity of the MjHKU4r-CoV-1 RBD for Malayan pangolin CD26 (0.30 ± 0.05 µM) was similar to that of hCD26 (0.26 ± 0.04 µM) (Fig. 5).AA sequence alignment for CD26s shows that Malayan pangolin CD26 has four AA distinctions participating in the interaction with the MjHKU4r-CoV-1 RBD compared to hCD26, which were V288, P289, T334, and S348 (Fig. 6A).Structural homology modeling showed that these AAs could not significantly influence the binding of CD26 to the hydrophobic pocket or the strong polar interactions between the MjHKU4r-CoV-1 RBD and CD26 interface (Fig. 7A,B).In addition, the binding capacity of the MjHKU4r-CoV-1 RBD for Pp bat CD26 (1.58 ± 0.06 µM) was lower than that of hCD26 (0.26 ± 0.04 µM) (Fig. 5).AA sequence alignment for CD26s showed that Pp bat CD26 has three AA distinctions participating in the interaction with the MjHKU4r-CoV-1 RBD compared to hCD26, which were T295, N334, and K336.SPR results demonstrated that T295 and K336 substitutions decrease binding affinity to the MjHKU4r-CoV-1 RBD (Fig. EV3).As observed in the cat CD26, T295 could influence the binding of CD26 to the hydrophobic pocket, and K336 may influence the H-bond with Y463 of the MjHKU4r-CoV-1 RBD (Fig. 7A,B).
Altogether, our study here confirms the strong binding of hCD26 with the MjHKU4r-CoV-1 RBD by biochemical and structural analyses.We also demonstrate that MjHKU4r-CoV-1 has a broad host range by binding affinity analysis, and we decipher the determinants of this host spectrum.All of these data point to the potential emergence of human/animal infections by MjHKU4r-CoV-1.

Gene cloning, protein production, and purification
The DNA sequences encoding the MjHKU4r-CoV-1 RBD (S residues E375-Y614, GWHBHAL01000000) and HKU4-CoV RBD The AA sequences of animal CD26s were renumbered based on hCD26 for convenient comparison.Interacting residues of hCD26 with the MjHKU4r-CoV-1 RBD are listed in the first column.The corresponding AAs in the CD26s from Tp bat, rat, hamster, cat, dog, and ferret are listed.The K D value for hCD26 bound to the MjHKU4r-CoV-1 RBD was 0.23 ± 0.09 µM, and that for each mutant is filled in in parentheses.K D values shown are the mean ± SD of at least three biological replicates.
(S residues E372-Y611, ABN10848.1)were separately cloned into a modified pFastbac Dual plasmid (Invitrogen) under the control of the polyhedrin promoter (Song et al, 2019).An N-terminal gp67 signal peptide and a C-terminal hexa-His tag were added to facilitate protein secretion and purification (Wang et al, 2014).Green fluorescent protein (GFP) was placed under the control of the P10 promoter to visualize its expression.Recombinant pFastbac Dual plasmid was transformed into DH10Bac-competent cells (Invitrogen, Cat# 10361-012) to produce the recombinant bacmid.
Transfection and virus amplification were conducted with Sf9 cells (Invitrogen, Cat# 11496015), and the recombinant proteins were expressed in High Five cells (Invitrogen, Cat# B85502) for 2 days.Sf9 and High Five cells were cultured in Insect-XPRESS™ Medium (Lonza, Cat# 12-730Q) and SIM HF Medium (Sino Biological, Cat# MHF1), respectively.Cell supernatant was collected, and soluble proteins were purified using HisTrap HP (Cytiva) and Superdex 200 Increase 10/300 GL (Cytiva) columns successively.DNA sequences encoding the MERS-RBD (S residues E367-Y606, AFS88936.1),hCD26 (residues S39-P766, NP_001926.2),and seven hCD26 mutants (hCD26-K267A, R317A, R336A, T288A, Q344A, and N229A/Q) fused to a C-terminal 6 × His tag were separately cloned into the pCAGGS vector.A Kozak sequence and an exogenous signal peptide derived from µ-phosphatase (MGILPSPGM-PALLSLVSLLSVLLMGCVAETGT) were added to the N-terminus to maximize protein production (Zhao et al, 2023).HEK293F cells (Gibco, Cat# 11625-019) were used to express these proteins.Cells were cultured in SMM 293-TII medium (Sino Biological, Cat# M293TII) with 5% CO 2 at 37 °C and 140 rpm and then were transfected with transfection reagent (Sino Biological, Cat# STF02) at a density of 2 × 10 6 cells/mL.Expression Medium (A) Hydrophobic interaction interface comparisons between hCD26 and CD26s from Tp bat, rat, hamster, cat, dog, and ferret.These six animal CD26 structures from structural homology modeling are aligned with the MjHKU4r-CoV-1 RBD/hCD26 complex.The MjHKU4r-CoV-1 RBD is shown as a surface according to hydrophobicity (green: hydrophilic; white: neutral; and gold: hydrophobic).The helix (residues 289-296) from hCD26 and corresponding regions on the animal CD26s are shown as cartoons and sticks and colored in green (human), orange (Tp bat), cyan (rat), cornflower blue (hamster), pink (cat), light gray (dog), and light coral (ferret).Differential residues between the two CD26s for comparison are labeled in their respective colors.(B) Polar interaction comparisons between hCD26 and CD26s from Tp bat, rat, hamster, cat, dog, and ferret.Residues 334-348 from hCD26 and corresponding regions on the animal CD26s are shown as cartoons, which are colored corresponding to (A).Interacting residues in the MjHKU4r-CoV-1 RBD/hCD26 complex are shown as sticks, as well as corresponding residues from animal CD26s.Differential residues between hCD26 and animal CD26 are labeled in their respective colors.

(
A) AA sequence alignment of the RBDs from HKU4-CoV, MjHKU4r-CoV-1, and MERS-CoV.(B) The SPR curves for HKU4-CoV, MjHKU4r-CoV-1, and MERS-CoV bound to hCD26 are shown.Raw curves are shown with different colors, as indicated.The fit curves are represented by black lines.K D values are the mean ± SD of three biological replicates.(C) The overall architecture of the MjHKU4r-CoV-1 RBD/hCD26 crystal structure.(D) Interaction network in the MjHKU4r-CoV-1 RBD/hCD26 complex.Side chains of interacting residues on hCD26 (green) and the MjHKU4r-CoV-1 RBD (hot pink) are shown as sticks and labeled appropriately.The red dashes present H-bonds.(E) Hydrophobic interactions between the MjHKU4r-CoV-1 RBD and hCD26.The MjHKU4r-CoV-1 RBD is shown as a surface according to hydrophobicity (green: hydrophilic; white: neutral; and gold: hydrophobic).The helix (residues 289-296) from hCD26 is shown as a cartoon and sticks and colored in green.Source data are available online for this figure.

Figure 2 .
Figure 2. Mutational analysis of key residues responsible for MjHKU4r-CoV-1 RBD binding.(A-F)SPR analysis of binding between the MjHKU4r-CoV-1 RBD and hCD26 (A) and its four mutants: hCD26-K267A (B), R317A (C), R336A (D), T288A (E), and Q344A (F).Raw curves are shown with different colors, as indicated.The fit curves are represented by black lines.K D values are the mean ± SD of three biological replicates.Source data are available online for this figure.

Figure 5 .
Figure 5.The binding affinities between CD26 orthologs and the MjHKU4r-CoV-1 RBD.SPR analyses of binding between the MjHKU4r-CoV-1 RBD and 18 CD26 orthologs covering eight orders were performed.Raw curves are shown with different colors, as indicated.The fit curves are represented by black lines.K D values are the mean ± SD of three biological replicates.Source data are available online for this figure.

Figure 6 .
Figure 6.Identification of host range determinants restricting MjHKU4r-CoV-1 recognition.(A) Part of the AA sequence alignment of the 18 CD26 orthologs from eight orders.Species that bind to the MjHKU4r-CoV-1 RBD are shaded in blue, otherwise in yellow.Residue sites on hCD26 interacting with the MjHKU4r-CoV-1 RBD are labeled using green triangles, and solid ones represent the residues participating in H-bonds.(B) SPR analysis of binding between the MjHKU4r-CoV-1 RBD and hCD26 and its seven mutants: hCD26-I295K, L294T, A291E, S292D, A291D, and S292A-I295T-R336K-Q344E. Raw curves are shown with different colors, as indicated.The fit curves are represented by black lines.K D values are the mean ± SD of three biological replicates.Source data are available online for this figure.

Figure EV1.
Figure EV1.Local density map of the interface between the MjHKU4r-CoV-1 RBD and hCD26.
Figure EV1.Local density map of the interface between the MjHKU4r-CoV-1 RBD and hCD26.The MjHKU4r-CoV-1 RBD and hCD26 are colored in hot pink and green, respectively.The local 2Fo-Fc map contoured at 0.5σ for its binding interface is shown as gray surfaces, and AAs are displayed as sticks.

Figure EV4 .
Figure EV4.Binding affinity measurement between the MjHKU4r-CoV-1 RBD and hCD26 using a capture method.Raw curves are shown in pink, and the fit curves are represented by black lines.K D values are the mean ± SD of three biological replicates.Source data are available online for this figure.

Table 1 .
X-ray data collection and refinement statistics.

Table 3 .
Key residue analysis for CD26s determining the host range of MjHKU4r-CoV-1.